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Identification of sex hormone binding globulin-interacting in the brain using phage display screening

M. GNANASEKAR1, F.G. SULEMAN2, K. RAMASWAMY1 and J.D. CALDWELL3

1Department of Biomedical Sciences, University of Illinois College of Medicine, 1601 Parkview Avenue, Rockford, IL 61107; 2Thermo Fisher Scientific, 3747 N. Meridian Road, Rockford, IL 61105; 3Department of Pharmacology, Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, USA

Received April 16, 2009; Accepted June 2, 2009

DOI: 10.3892/ijmm_00000248

Abstract. The present study reports the identification of Nakhla et al (2,3) demonstrated that SHBG binds to a receptor human sex hormone binding globulin (SHBG)-interacting or receptor complex which then results in increases of intra- proteins in the brain using a phage display-based screening cellular cAMP. Studies by Rosner and colleagues (3,4) suggest technology. Phage display is a system in which a foreign that the putative SHBG receptor is a G -coupled receptor protein is displayed on the surface of a bacteriophage as a or functionally linked to one. In these studies (1) a 167-kDa fusion protein with one of the coat proteins of the bacteriophage. protein was solubilized from membranes of human prostate T7 phage clones expressing normal human brain proteins glands and its binding activity to SHBG was demonstrated (human normal brain phage-display cDNA expression library) using 125I-SHBG. Other studies (5,6) also suggested that there were screened using SHBG as bait. The bound phage clones are membrane-associated receptors for SHBG in the uterus. were then identified by DNA sequencing and by BLAST These membrane-associated receptors may be important for search analysis. Of the twenty binding proteins analyzed, three internalization of SHBG, which was observed in many tissues were found to be membrane-associated proteins: synaptosomal (7-14) including the brain (15). The Nykjaer and Willnow associated protein 25 (SNAP25), Thy-1 cell surface antigen and laboratory demonstrated that a membrane-associated protein zonadhesin. Further studies will determine if the interactions of called megalin, a 600-kDa member of the LDL receptor SHBG with these proteins have any role in the internalization family, is capable of binding to and facilitating the internalization and cell signaling events or whether they contribute to steroid of both SHBG and its bound steroid (16). It is the protein that delivery to specific cells. the same laboratory previously suggested was involved in the uptake of other steroids and demonstrated that it belonged to Introduction a larger class of proteins responsible for the uptake of a broad range of lipophilic molecules (17). Megalin is important in Examining the roles of steroid binding globulins as active steroid effects. This group demonstrated that without megalin, participants in steroid actions has received attention recently. mice showed a delayed onset of puberty (16). The same One reason is that there are membrane-associated receptors laboratory demonstrated the importance of binding globulins for binding globulins. Hryb et al (1) showed that sex hormone in mediating the effects of steroids. Mice lacking a cortico- binding globulin (SHBG) binds with high affinity to a specific steroid-binding globulin, another steroid-binding globulin membrane receptor in prostate stromal and epithelial cells. found in the brain (18,19), had more free steroids in the blood but showed fewer physiological responses to them, suggesting that the presence of this binding globulin was necessary in ______order to see the effects of corticosteroids (20). The first indication that there were SHBG receptors in the Correspondence to: Dr Munirathinam Gnanasekar, Department brain was when SHBG facilitated female sexual receptivity (21) of Biomedical Sciences, University of Illinois College of Medicine, except when coupled to dihydrotestosterone (22). Our laboratory 1601 Parkview Avenue, Rockford, IL 61107, USA and others also found that SHBG is produced in the brain, E-mail: [email protected] where it is often co-localized with the neuropeptides oxytocin Dr Jack D. Caldwell, Department of Pharmacology, Lake Erie and vasopressin (23-25). Since both SHBG and oxytocin are College of Osteopathic Medicine, 1858 West Grandview Boulevard, often found together in synaptic vesicles in axonal varicosities, Erie, PA 16509, USA we concluded that SHBG is likely released into the brain. E-mail: [email protected] This suggests the existence of receptors for this released SHBG. We also presented indirect evidence of such SHBG Key words: sex hormone binding globulin, brain, phage display, receptors in the hypothalamus (26). After binding to membrane- zonadhesin, synaptosomal associated protein 25 associated receptors, it is possible that SHBG, its associated steroid, and the SHBG receptor are all internalized into brain cells. Caldwell et al (15) demonstrated uptake of SHBG in vivo Gnanasekar 16/04 14/8/2009 12:23 ÌÌ Page 422

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Figure 2. Size distribution of phage clones obtained from first round of biopanning. Phage bound to SHBG from the first cycle of biopanning were eluted and amplified on BLT5403. The amplified phages were plated with BLT5403 on LB agar plates for plaque formation. Ten plaques were randomly selected for PCR amplification. Amplified PCR products (5 μl) were loaded on 1% agarose gel containing Etbr and electrophoresed for 30 min. The resolved gel was observed on a UV transilluminator and the image was captured using Kodak digital software. Lane 1, control PCR that lacks a Figure 1. Schematic strategy to identify targets of SHBG. template; lanes 2-11 random phage clones.

in the choroids plexus, paraventricular and supraoptic nuclei, and in specific periventricular cells. Despite observations that of the boiled samples were used as DNA templates for PCR suggest the presence of both central and peripheral SHBG amplification. membrane receptors, the identity of brain SHBG receptors has not been elucidated. Another study utilized a yeast two-hybrid PCR and DNA sequencing. DNAs of the selected phage clones system to identify candidate proteins for SHBG binding in were amplified by PCR according to the manufacturer's prostate (27). In this study, phage display of a human brain (Novagen) instructions on a Perkin Elmer Thermocycler cDNA library is used to identify SHBG-interacting proteins. using the T7Select up 5'-GGAGCTGTCGTATTCCAGTC-3' and T7Select down 5'-GGCTGATACCACCCTTCAAG-3' as Materials and methods forward and reverse primers respectively. PCR conditions were 30 cycles of denaturation at 94˚C for 30 sec, 55˚C annealing Materials. The normal human brain T7Select™ phage library, for 30 sec, and 72˚C extension for 30 sec. A final extension at host strain BLT5403, as well as primers were obtained from 72˚C was for 5 min. The reaction products were resolved on Novagen, USA. Qiaquick PCR purification kits were from agarose gel to confirm the presence of amplification products. Qiagen (Valencia, CA). Jumpstart TAQ polymerase was from The PCR products were then purified using the Qiaquick Sigma (St. Louis, MO). PCR purification kit (Qiagen, Valencia, CA) and the inserts were sequenced on both strands at DNA Services Facility Biopanning. The strategy to identify the SHBG interacting (Research Resources Center, University of Illinois at Chicago, proteins is presented in Fig. 1. Briefly, biopanning was IL) to confirm the authenticity of the . The determined performed as follows. SHBG was diluted in deionized water to DNA sequences were then analyzed by BLAST analyses. 10 μg/ml; 100 μl aliquots were coated in the wells of a 96-well microtiter plate (Costar, Corning, NY) and incubated at room Results temperature (RT) for 3 h. The SHBG-coated wells were then washed with Tris-buffered saline (TBS), blocked by adding Over twenty phage clones bound to SHBG. Of these, initially 200 μl 3% BSA to each well and incubated at RT for 1 h. we isolated ten phage plaques from the fourth round of bio- Excess blocking reagent was removed by washing with panning and processed them for sequencing. Agarose gel deionized water. The T7Select phage of the human normal resolution of the PCR products showed that they were all the brain cDNA library (Novagen, USA) was added to wells and same size (data not shown), so we sequenced two clones out of incubated at RT for 1 h. Unbound phages were washed off the ten. Sequence analysis demonstrated that SHBG-interacting with TBS containing Tween while bound phages were then phage clones encoded for FAU gene products [ubiquitin-like eluted by incubating each well with 200 μl of 1% SDS in protein (FUBI) and ribosomal protein S30] (28). TBS. The eluted phages were then used to infect E. coli Because of the low molecular weight of these two candidate BLT5403 which amplifies the phages for the next biopanning proteins, we decided to screen more plaques from the first cycle. Three more biopanning rounds were done to increase biopanning. Through this process we isolated twenty-four the phage binding affinity to the SHBG. Finally, the phages more phage clones. All these clones were then processed for obtained after this fourth amplification were plaque titered sequencing. Fig. 2 shows the size distribution of the phage with a series of dilutions (10-2 to 10-14) on LB agar plates clones that were obtained after PCR amplification. The size containing 1% agarose and incubated for 2-4 h at 37˚C. Single of these clones varied between 570 and 1,265 bases. Each of plaques of phages were randomly selected with a pipette tip, the sequences was then subjected to a BLAST search to resuspended in 100 μl of 0.1X TE buffer, boiled for 10 min determine their identity. Table I shows the list of candidate and placed on ice for 5 min. To amplify the phage DNA, 1 μl proteins encoded by a phage that bound to SHBG. These Gnanasekar 16/04 14/8/2009 12:23 ÌÌ Page 423

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Table I. Putative proteins that bind to SHBG by phage display that interact with SHBG. This technique identified over twenty technique. binding proteins. Of these identified candidate proteins three ––––––––––––––––––––––––––––––––––––––––––––––––– are localized to the [SNAP25 (35), Thy-1 cell Phage clone no. Encoding proteins surface antigen (36), and zonadhesin (37)]. Some of the other ––––––––––––––––––––––––––––––––––––––––––––––––– proteins include GTP-binding protein (RAB3A) (38), myelin Membrane basic protein (39) and vacuolar protein sorting factor 4A (40). 1 Synaptosomal-associated protein These findings demonstrate that both cytoplasmic and (SNAP25), 25 kDa membrane proteins interact with SHBG. The majority of the 2 Thy-1 glycoprotein, Thy-1 cell identified proteins are intracellular with a good number of surface antigen them involved in protein synthesis. 3 Zonadhesin (ZAN) Pope and Lee sequenced at least sixty proteins from over 230 interacting proteins they obtained using the yeast two- Dual localization: hybrid system to identify prostatic SHBG binding proteins Membrane and intracellular (27). Two of the metabolic cytosolic proteins we identified, 4 GTP-binding protein (RAB3A) Adenylate kinase 5 and Phospolipase A2, group IVC, were 5 Myelin basic protein also identified by them (27). Adenylate kinase is involved in 6 Vacuolar protein sorting factor 4A the homeostasis of adenine and guanine nucleotides (41), while Intracellular phospholipase A2 catalyzes the hydrolysis of the 2-acyl bond 7 Adenylate kinase 5 of 3-n-phosphoglycerides (42). However, despite the high 8 ADP-ribosylation factor 6 number of membrane proteins they obtained, none of them interacting protein 4 (ARL61P4) are high molecular weight proteins. Interestingly, adenylate kinase and phospholipase A2 were shown to be over-expressed 9 Cystatin C in prostatic cancer (43,44) and the putative SHBG receptor 10 Cytoplasmic linker 2 (CYLN2), was also first described in the prostate (1). Given the variant 2 association of these proteins with SHBG in the brain it is not 11 Fatty acid binding protein 5 known at this time if these proteins are also involved in brain 12 Fubi (also coded for by the FAU gene) cancer. If so, elucidation of the interaction of SHBG with 13 FUS glycin rich protein these proteins may have implications for steroid-initiated 14 Lymphoid-enhancer binding factor1 tumorigenesis. 15 Mitochondrial ribosomal protein S6 Because others have postulated that the SHBG receptor is 16 Phospolipase A2, group IVC associated with the cellular membrane, we will first discuss (cytosolic calcium-independent) candidate proteins associated with the cellular membrane. Of (PLA2G4C) gene the three membrane-associated candidate proteins, only 17 PR-domain zinc finger protein 2 variant zonadhesin has a molecular weight that corresponds to the molecular weight suggested by Hryb et al (1) for the putative 18 Ribosmal protein L15 SHBG receptor, i.e. 167 kDa. It is known that zonadhesin 19 Ribosomal protein L10 forms two disulfide-bonded monomeric constituents of 105 20 Ribosomal protein L13 and 45 kDa, for a total molecular weight of 150 kDa. 21 Ribosomal protein L17, transcript 1 Zonadhesin is a mosaic protein in sperm membrane fractions 22 Ribosomal protein S30 (FAU), that binds directly and in a species-specific manner to the 23 elongation factor A extracellular matrix (zona pellucida; ZP) of the oocytes. Post- (SII)-like 3 (TCEAL3) translational processing leads to multiple forms of zonadhesin 24 Zinc finger protein 260 with differing apparent avidities for the ZP (45). In the pig, ––––––––––––––––––––––––––––––––––––––––––––––––– the p105/45 form binds preferentially to conspecific ZP (45). It is interesting that with the discovery of membrane receptors for progesterone in oocytes (46-48), that rapid steroid effects include cell membrane associated proteins (SNAP25, Thy-1 in oocytes are receiving renewed interest. We found SHBG cell surface antigen, and zonadhesin), GTP-binding protein immunoreactivity in prostatic secretions (24), which suggests (RAB3A), myelin basic protein and vacuolar protein sorting that SHBG in the ejaculate serves as a stimulatory agent at factor 4A. oocyte membrane receptors, as with zonadhesin. However, it is not known whether zonadhesin exists in the brain. Using Discussion Northern blot analysis, zonadhesin expression was found only within the testes and it was not detected in other organs SHBG is a 94-kDa protein found in the blood (10,29,30), brain including the brains of mice (49). However, due to the presence (23-25), and the male reproductive tract (24,31). One model of of MAM, mucin, D-, and EGF domains in zonadhesin, Gao and SHBG action suggests that it is a passive carrier of steroids in Garbers (49) suggested that apart from binding to zona blood, while another model suggests that there are receptors pellucida, mouse zonadhesin functions in other cell adhesion for SHBG on cellular membranes that mediate intracellular processes. Our suggestion that zonadhesin exists in the human actions (1,5,21,22,32-34). In this study, we tested whether we brain might indicate its involvement in cell adhesion and could utilize the phage display technique to identify proteins possibly cell migration during development. Gnanasekar 16/04 14/8/2009 12:23 ÌÌ Page 424

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Another indicator that a candidate protein is associated plasma membrane of murine T-lymphoma cells (59). To our with the membrane is coupling to G proteins. We identified knowledge, no such activity has been demonstrated in brain RAB3A as a candidate protein, which is a GTP-binding cells. In any case, the low molecular weight of Thy-1 protein of 24 kDa which belongs to the GTP-binding protein suggests that it could only be part of a large receptor complex superfamily. RAB3A and its interacting proteins are involved for SHBG. in Ca2+-dependent exocytosis (50). RAB3A has been identified Another candidate protein was vacuolar protein sorting in membrane and cytosolic fractions of mouse with the pre- factor 4A (VPS4), which is a member of the ATPases dominant form being membrane-bound. The localization to Associated with diverse cellular Activities (AAA) ATPase acrosomal membrane suggests a role for RAB3A in the family and a central regulator for early endosome trafficking regulation of zona pellucida-induced acrosomal exocytosis (60). This protein is well characterized in yeast where it is (51) that, along with identification of zonadhesin, may indicate known to bind to oxysterol binding protein and regulate a parallel receptor complex in brain and acrosome. intracellular lipid and vesicular transport (25). Since VPS4 Soluble N-ethylmaleimide-sensitive factor attachment has a molecular weight of 48 kDa (61), it is not likely to be the protein (SNAP-25) is an integral membrane protein localized anticipated SHBG receptor, however it could be one of the to the plasma membrane of pre-synaptic axon terminals. As a components of the receptor, which after being internalized, component of the SNARE (receptor for SNAPs), SNAP-25 functions in the transport of SHBG-bound steroid. appears to play an important role in the docking and fusion of Although FUBI and ribosomal protein S30 are cytosolic pre-synaptic vesicles during exocytosis of neurotransmitters low molecular weight proteins, they were obtained at the (52). Pope and Lee (27) identified another protein called fourth panning suggesting that they had the highest affinity SNARE associated protein which is also a component of the for SHBG. FUBI is synthesized with ribosomal S30 as its C- SNARE complex (53). SHBG internalization was observed in terminal extension. It was proposed that the fusion protein is a number of tissues (7,8,10,11,13,54) including the brain (15), post-translationally processed to generate free FUBI and free so it is possible that some of the proteins we identified here, in ribosomal protein S30. FUBI is a member of the ubiquitin particular SNAP-25, are involved in that internalization process. family, and ribosomal protein S30 belongs to the S30E family We also discovered that one protein binding to SHBG is of ribosomal proteins. Whereas the function of FUBI is myelin basic protein. Myelin basic protein (MBP) makes up currently unknown, ribosomal protein S30 is a component of ~30% of the components of a myelin sheath which electrically the 40S subunit of the cytoplasmic ribosome (62). It is also insulates axons and aids conduction of nerve impulses. There interesting to note the high number of other ribosomal is significant interest in MBP because of its role in demyelinating proteins that are involved in binding to SHBG. It is not diseases, particularly multiple sclerosis (MS). Several studies known what the consequences of the interaction of SHBG have shown a role for against MBP in the patho- with FUBI or ribosomal proteins are or any possible genesis of MS (55). It is possible that one function of SHBG involvement in steroid metabolism. However, it will be in the brain is due to actions at the level of myelin in brain interesting to investigate, using cell culture systems, how fiber tracts. SHBG internalization was often associated with these proteins are modified in the presence of SHBG and such tracts in the brain (15). It is also interesting to note that how their deficiencies affect steroid metabolism. MBP is present in various different cell types including This phage display system did not identify megalin which lymphoid cells and all types of myeloid lineage cells (i.e., is a candidate for the SHBG receptor as mentioned above. macrophages, dendritic cells, granulocytes, megakaryocytes, The failure of SHBG to identify megalin may be due to the and erythroblasts) (56). At least five isoforms of MBP were inability of phages to fuse large proteins or simply that identified with molecular weights ranging from 14 to 21 kDa. megalin is absent in the brain. (57). Again, this is not likely the SHBG receptor, but it could Overall, it seems likely that the SHBG receptor is made be a component of it. It was demonstrated that supplemental up of several protein subunits and that any one of the proteins estrogen enhances the efficacy of TCR-based immunotherapy we identified could be a component of the large molecule. For for autoimmune diseases that are predominant in females (58). example, there are four proteins in the presynaptic terminal Estrogen potentiates treatment with T-cell-receptor protein of involved in neurotransmitter release, RAB3A, SNAP-25, female mice with experimental encephalomyelitis (EAE). This VAMP/-2 and , all form a complex of is a significant finding because the involvement of estrogen in ~119 kDa (63). Another study that lends credibility to this the therapy of EAE (a mouse model for MS) also suggests a argument is by Oike et al (64) who showed that Group IIA role for SHBG in its transportation and hence potential phospholipase A2 is coexpressed with SNAP-25 in rat mature therapy for MS. taste receptor cells that possess exocytotic function. If several Thy-1 glycoproteins were identified as candidate proteins elements of the SHBG receptor complex are only transiently as well. Thy-1 glycoproteins are constituents of thymocytes found in the membrane, it may explain why Pope and Lee (27) and neurons. Thy-1 cell surface antigen is a small protein were unable to identify a single protein of high molecular (18 kDa) that may play a role in cell-cell or -ligand interactions weight using their yeast two-hybrid method. during synaptogenesis and other events in the brain (UniProtKB/ Because the phages of the Phage Display technique display Swiss-Prot, Institute of Bioinformatics and the EMBL outstation- all proteins on their surface, it may be that we here demonstrated the European Bioinformatics Institute). It was also shown that SHBG binding to proteins that are normally only able to despite the fact that Thy-1 glycoprotein is anchored (glycosyl- interact with SHBG within the cell. SHBG internalization has phosphatidal-insoitol anchor) to the outer hemi-leaflet of the been demonstrated in multiple tissues (7-9,11-13,54) including cell membrane, it is capable of transducing signals across the the brain (15) suggesting that SHBG along with associated Gnanasekar 16/04 14/8/2009 12:23 ÌÌ Page 425

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